Showing papers in "Biochemical Journal in 1995"

Capacitative calcium entry.

Abstract: The original idea that calcium might enter cells through a capacitative mechanism was first introduced by Jim Putney [1]. The somewhat surprising hypothesis was that calcium entry was regulated by the state of filling of the calcium stores. By analogy with a capacitor in an electrical circuit, the calcium stores prevent entry when they are charged up but immediately begin to promote entry as soon as stored calcium is discharged. This capacitative entry mechanism is present in many cells and has properties which are very similar from one cell to the next [2-6]. Capacitative calcium entry can be switched on by a great variety of stimuli such as normal agonists or pharmacological agents all of which share a common property of releasing stored calcium. Examples include calcium-mobilizing agonists [7-13], the calcium-mobilizing second messenger inositol 1,4,5trisphosphate (InsP3) [11,12,14-18], the calcium ionophore ionomycin [14,19], inhibitors of the endoplasmic reticulum (ER) pumps such as thapsigargin [8,10,12,20] and cyclopiazonic acid [20,21] or simply by incubating cells in Ca2+-free conditions [12,14,15]. All this evidence indicates that the entry of external calcium is somehow controlled by the calcium content of the ER. This review is focused primarily on the mechanism ofcapacitative Ca2+ entry.

Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion.

Abstract: 1. The yield of mitochondria isolated from perfused hearts subjected to 30 min ischaemia followed by 15 min reperfusion was significantly less than that for control hearts, and this was associated with a decrease in the rates of ADP-stimulated respiration. 2. The presence of 0.2 microM cyclosporin A (CsA) in the perfusion medium during ischaemia and reperfusion caused mitochondrial recovery to return to control values, but did not reverse the inhibition of respiration. 3. A technique has been devised to investigate whether the Ca(2+)-induced non-specific pore of the mitochondrial inner membrane opens during ischaemia and/or reperfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[3H]glucose ([3H]DOG), which will only enter mitochondria when the pore opens. Subsequent isolation of mitochondria demonstrated that [3H]DOG did not enter mitochondria during global isothermic ischaemia, but did enter during the reperfusion period. 4. The amount of [3H]DOG that entered mitochondria increased with the time of ischaemia, and reached a maximal value after 30-40 min of ischaemia. 5. CsA at 0.2 microM did not prevent [3H]DOG becoming associated with the mitochondria, but rather increased it; this was despite CsA having a protective effect on heart function similar to that shown previously [Griffiths and Halestrap (1993) J. Mol. Cell. Cardiol. 25, 1461-1469]. 6. The non-immunosuppressive CsA analogue [MeAla6]cyclosporin was shown to have a similar Ki to CsA on purified mitochondrial peptidyl-prolyl cis-trans-isomerase and mitochondrial pore opening, and also to have a similar protective effect against reperfusion injury. 7. Using isolated heart mitochondria, it was demonstrated that pore opening could become CsA-insensitive under conditions of adenine nucleotide depletion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [3H]DOG in the CsA-perfused hearts is explained by the ability of the drug to stabilize pore closure and so decrease the loss of [3H]DOG from the mitochondria during their preparation.

A hierarchy of ATP-consuming processes in mammalian cells.

Abstract: The rates of different ATP-consuming reactions were measured in concanavalin A-stimulated thymocytes, a model system in which more than 80% of the ATP consumption can be accounted for. There was a clear hierarchy of the responses of different energy-consuming reactions to changes in energy supply: pathways of macromolecule biosynthesis (protein synthesis and RNA/DNA synthesis) were most sensitive to energy supply, followed by sodium cycling and then calcium cycling across the plasma membrane. Mitochondrial proton leak was the least sensitive to energy supply. Control analysis was used to quantify the relative control over ATP production exerted by the individual groups of ATP-consuming reactions. Control was widely shared; no block of reactions had more than one-third of the control. A fuller control analysis showed that there appeared to be a hierarchy of control over the flux through ATP: protein synthesis > RNA/DNA synthesis and substrate oxidation > Na+ cycling and Ca2+ cycling > other ATP consumers and mitochondrial proton leak. Control analysis also indicated that there was significant control over the rates of individual ATP consumers by energy supply. Each ATP consumer had strong control over its own rate but very little control over the rates of the other ATP consumers.

Serine/threonine protein phosphatases

Abstract: 1 (1-1) and inhibitor-2 (1-2), and preferentially dephosphorylate the /3-subunit of phosphorylase kinase, whereas type-2 phosphatases are insensitive to the heat-stable inhibitors and preferentially dephosphorylate the x-subunit of phosphorylase kinase [1,2]. Type-2 phosphatases can be further subdivided into spontaneously active (PP2A), Ca2l-dependent (PP2B) and Mg2+dependent (PP2C) classes. The use of okadaic acid, a specific phosphatase inhibitor, further facilitated the discrimination between different classes [3]. Although still widely in use, this classificatioh does not reflect the actual phylogenetic relationship between the different Ser/Thr protein phosphatases. Molecular cloning revealed that PP2A was in fact much more closely related to PPI than to PP2C. Moreover, in the past few years many novel protein phosphatases have been identified that do not fit into the above classification. Many of these protein phosphatases are closely related to the existing classes or are intermediates between classes. From a phylogenetic point of view, it would be more reasonable to group PP 1, PP2A and PP2B in a family I and PP2C in a family II. Recently a detailed comparison of the primary structures of 44 different protein Ser/Thr phosphatases, excluding PP2C, was carried out [4]. This revealed a common core structure that comprises two domains. The first domain is predicted to fold as

Cyclins and cyclin-dependent kinases: a biochemical view

Abstract: Cyclins are the activating partners of a highly conserved family of protein kinases, the cyclin-dependent kinases (CDKs). A number of diverse cyclins and CDKs are now known, many of which play important roles in the regulation of the eukaryotic cell cycle. However, it has recently become clear that the cyclin-CDK motif is used by the cell to control processes separate from the cell cycle, such as the response to phosphate starvation in yeast. This may be because the cyclin-CDK motif offers a remarkable degree of flexibility in response to variations in the environment. Such flexibility is conferred by the ability to alter the activity ofthe cyclin-CDK complex by phosphorylation, or by binding specific inhibitor proteins, or by varying the level of the cyclin itself. In this review I will outline the mechanisms by which cyclin-CDK activity can be modulated, and how particular aspects of this regulation are more important in some cell cycle events compared with others. Given the limitations of space I will not give a detailed review of cell cycle regulation. Readers who would like an overview of the cell cycle are directed to a number of recent reviews [1-7].

Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1

Abstract: The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.

Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase

Abstract: The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo

Abstract: Albumin is a multifunctional transport protein that binds a wide variety of endogenous substances and drugs. Insulins with affinity for albumin were engineered by acylation of the epsilon-amino group of LysB29 with saturated fatty acids containing 10-16 carbon atoms. The association constants for binding of the fatty acid acylated insulins to human albumin are in the order of 10(4)-10(5) M-1. The binding apparently involves both non-polar and ionic interactions with the protein. The acylated insulins bind at the long-chain fatty acid binding sites, but the binding affinity is lower than that of the free fatty acids and depends to a relatively small degree on the number of carbon atoms in the fatty acid chain. Differences in affinity of the acylated insulins for albumin are reflected in the relative timing of the blood-glucose-lowering effect after subcutaneous injection into rabbits. The acylated insulins provide a breakthrough in the search for soluble, prolonged-action insulin preparations for basal delivery of the hormone to the diabetic patient. We conclude that the biochemical concept of albumin binding can be applied to protract the effect of insulin, and suggest that derivatization with albumin-binding ligands could be generally applicable to prolong the action profile of peptide drugs.

Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide

Abstract: We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM). Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore ('megachannel') remained closed continuously. After addition of 100 microM t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was accompanied by mitochondrial depolarization, as shown by loss of TMRM. Subsequently, the concentration of ATP declined and cells lost viability. Trifluoperazine, a phospholipase inhibitor that inhibits the permeability transition in isolated mitochondria, prevented calcein redistribution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Trifluoperazine did not prevent ATP depletion and cell death after the addition of CCCP. In conclusion, the permeability transition pore does not 'flicker' open during normal incubation of hepatocytes but remains continuously closed. Moreover, mitochondrial depolarization per se does not cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.

The gabaa receptors

Abstract: INTRODUCTION y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. It mediates its effects via the specific interaction with the integral membrane proteins, the GABA receptors. GABA receptors can be classified according to their respective transduction mechanisms following activation, as the GABA and the GABAB receptor respectively. The focus of this review is the GABAA receptor. GABAA receptors are fastacting ligand-gated chloride ion channels. Thus receptor activation in the brain is followed within milliseconds by the gating

Integrin alpha 2 beta 1-independent activation of platelets by simple collagen-like peptides: collagen tertiary (triple-helical) and quaternary (polymeric) structures are sufficient alone for alpha 2 beta 1-independent platelet reactivity.

Abstract: The platelet reactivities of two simple collagen-like synthetic peptides, Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly and Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly, were investigated. Both peptides adopted a stable triple-helical conformation in solution. Following cross-linking, both peptides proved to be highly platelet-aggregatory, more active than collagen fibres, inducing aggregation at concentrations as low as 20 ng/ml. These peptides formed microaggregates in solution, and cross-linking was thought to stabilize these structures, allowing expression of their platelet reactivity at 37 degrees C. Like collagen fibres, the peptides caused platelet secretion and release of arachidonate from platelet membrane lipids as well as activation of integrin alpha IIb beta 3 culminating in aggregation. Monoclonal antibodies directed against the integrin alpha 2 beta 1 failed to prevent aggregation release of arachidonate or platelet adhesion to the peptides. Our results indicate that collagen can activate platelets by a mechanism that is independent of integrin alpha 2 beta 1 and for which collagen tertiary and quaternary structures are sufficient alone for activity without the involvement of highly specific cell-recognition sequences.

Lysophospholipids activate ovarian and breast cancer cells

Abstract: We have investigated the effects of phospholipids on activation and proliferation of ovarian and breast cancer cells. Lysophosphatidic acid (LPA), lysophosphatidylserine (LPS) and sphingosylphosphorylcholine (SPC) all induce transient increases in cytosolic free Ca2+ ([Ca2+]i) in both ovarian and breast cancer cell lines. The ability of LPA, LPS and SPC to induce increases in [Ca2+]i in ovarian and breast cancer cells is likely to be due to an interaction with cell-surface receptors as the increases in [Ca2+]i were: (1) due to release of calcium from intracellular stores and not from transmembrane uptake due to changes in permeability; (2) blocked by lanthanum and suramin which do not enter cells; (3) blocked by phorbol esters which interrupt increases in [Ca2+]i induced through a number of different receptors; and (4) not detected in freshly isolated peripheral blood mononuclear cells, indicating cell type specificity. In addition, increases in [Ca2+]i induced by LPA, LPS and SPC in ovarian and breast cancer cells completely self-desensitized and cross-desensitized each other, but did not block increases in [Ca2+]i induced by thrombin. Lysophosphatidylglycerol (LPG), but not other lysophospholipids, inhibited LPA- but not LPS- or SPC-induced increases in [Ca2+]i, suggesting that LPA may interact with a different receptor(s) to LPS or SPC and that their downstream signalling pathways converge or interact. LPA, SPC and LPS also induced rapid increases in tyrosine phosphorylation of specific cellular proteins, including p125FAK. Strikingly, LPA, but not LPS or SPC, induced activation of mitogen-activated protein (MAP) kinases. Despite an ability to activate similar intracellular signaling events, LPA, LPS and SPC exhibited markedly different effects on cell proliferation. Whereas LPA induced a significant increase in cell proliferation, LPS did not substantially alter cell proliferation and SPC inhibited cell proliferation. Surprisingly, phosphatidic acid (PA), which did not induce increases in [Ca2+]i, p125FAK activation or activation of MAP kinases, did induce proliferation of ovarian cancer cells, albeit at higher concentrations that LPA. The discordance between sensitivity to LPG, early biochemical events stimulated, and the eventual proliferation response combine to suggest that LPA probably utilizes a different receptor from LPS, SPC and PA. Therefore ovarian and breast cancer cells are sensitive to the effects of a number of different phospholipids which may play a role in the growth of these tumour cells in the cancer patient and are thus potential targets for therapy.

Acute cold-induced suppression of ob (obese) gene expression in white adipose tissue of mice: mediation by the sympathetic system

Abstract: The effect of acute exposure to cold on the expression of the ob (obese) gene, which encodes a protein that plays a critical role in the regulation of energy balance and body weight, has been examined in epididymal white adipose tissue of mice. Overnight (18 h) exposure of mice to a temperature of 4 degrees C led to the disappearance of ob mRNA in epididymal white fat, and subsequent studies showed that a cold-induced loss of ob mRNA could occur in as little as 2-4 h of exposure to 4 degrees C. When mice exposed to cold for 18 h were returned to the warm (24 degrees C), there was a rapid stimulation of the expression of the ob gene, the mRNA returning within 2.5 h. Administration of noradrenaline led to a reduction in the level of ob mRNA in mice maintained in the warm, while isoprenaline resulted in the disappearance of the mRNA; these changes in ob mRNA were paralleled by similar changes in lipoprotein lipase mRNA. In contrast to white fat, the level of lipoprotein lipase mRNA in brown adipose tissue was increased by noradrenaline and isoprenaline. It is concluded that there is a cold-induced suppression of ob gene expression in white adipose tissue of mice and that this is mediated primarily by the sympathetic system. The profound effect of cold on ob gene expression indicates that the ob system relates to energy expenditure, as well as to satiety.

Increase in levels of polyubiquitin and proteasome mRNA in skeletal muscle during starvation and denervation atrophy.

Abstract: Most of the increased protein degradation in muscle atrophy caused by starvation and denervation is due to activation of a non-lysosomal ATP-dependent proteolytic process. To determine whether expression of the ubiquitin-proteasome-dependent pathway is activated in atrophying muscles, we measured the levels of mRNA for ubiquitin (Ub) and proteasome subunits, and Ub content. After rats had been deprived of food for 1 or 2 days, the concentration of the two polyubiquitin (polyUb) transcripts increased 2-4-fold in the pale extensor digitorum longus muscle and 1-2.5-fold in the red soleus, whereas total muscle RNA and total mRNA content fell by 50%. After denervation of the soleus, there was a progressive 2-3-fold increase in polyUb mRNA for 1-3 days, whereas total RNA content fell. On starvation or denervation, Ub concentration in the muscles also rose by 60-90%. During starvation, polyUb mRNA levels also increased in heart, but not in liver, kidney, spleen, fat, brain or testes. Although the polyUb gene is a heat-shock gene that is induced in muscles under certain stressful conditions, the muscles of starving rats or after denervation did not express other heat-shock genes. On starvation or denervation, mRNA for several proteasome subunits (C-1, C-3, C-5, C-8 and C-9) also increased 2-4-fold in the atrophying muscles. When the food-deprived animals were re-fed, levels of Ub and proteasome mRNA in their muscles returned to control values within 1 day. In contrast, no change occurred in the levels of muscle mRNAs encoding cathepsin L, cathepsin D and calpain 1 on denervation or food deprivation. Thus polyUb and proteasome mRNAs increased in atrophying muscles in co-ordination with activation of the ATP-dependent proteolytic process.

Tissue-specific regulation of selenoenzyme gene expression during selenium deficiency in rats.

Abstract: Regulation of synthesis of the selenoenzymes cytosolic glutathione peroxidase (GSH-Px), phospholipid hydroperoxide glutathione peroxidase (PHGSH-Px) and type-1 iodothyronine 5'-deiodinase (5'IDI) was investigated in liver, thyroid and heart of rats fed on diets containing 0.405, 0.104 (Se-adequate), 0.052, 0.024 or 0.003 mg of Se/kg. Severe Se deficiency (0.003 mg of Se/kg) caused almost total loss of GSH-Px activity and mRNA in liver and heart. 5'IDI activity decreased by 95% in liver and its mRNA by 50%; in the thyroid, activity increased by 15% and mRNA by 95%. PHGSH-Px activity was reduced by 75% in the liver and 60% in the heart but mRNA levels were unchanged; in the thyroid, PHGSH-Px activity was unaffected by Se depletion but its mRNA increased by 52%. Thus there is differential regulation of the three mRNAs and subsequent protein synthesis within and between organs, suggesting both that mechanisms exist to channel Se for synthesis of a particular enzyme and that there is tissue-specific regulation of selenoenzyme mRNAs. During Se depletion, the levels of selenoenzyme mRNA did not necessarily parallel the changes in enzyme activity, suggesting a distinct mechanism for regulating mRNA levels. Nuclear run-off assays with isolated liver nuclei showed severe Se deficiency to have no effect on transcription of the three genes, suggesting that there is post-transcriptional control of the three selenoenzymes, probably involving regulation of mRNA stability.

Binding of gelatinases A and B to type-I collagen and other matrix components.

Abstract: Matrix sequestration of matrix metalloproteinases may be important for the facilitation of remodelling events and the migration of cells through the extracellular matrix. Using an ELISA technique we studied the ability of pro and active forms of gelatinases A and B (GLA and GLB) to bind to matrix components and the contribution made by the different enzyme domains. Pro and active forms of GLA and GLB bound to type-I and type-IV collagens, gelatin and laminin films. Binding to collagens occurred exclusively via the N-terminal portion of the molecule in both of the gelatinases; deletion of the fibronectin-like domain in GLA abolished binding. Fibronectin was shown to compete with GLA, confirming that binding occurs through this domain. GLA and GLB competed for binding to collagen type I, whereas collagenase and stromelysin bound to different sites and could be co-localized with the gelatinases. We conclude that gelatinases have different binding specificities from those previously documented for stromelysin and collagenase, which bind through their C-terminal domains to collagen fibrils.

Fibrillogenesis in Alzheimer's disease of amyloid beta peptides and apolipoprotein E.

Abstract: A central event in Alzheimer's disease is the conformational change from normally circulating soluble amyloid beta peptides (A beta) and tau proteins into amyloid fibrils, in the form of senile plaques and neurofibrillary tangles respectively. The apolipoprotein E (apoE) gene locus has recently been associated with late-onset Alzheimer's disease. It is not know whether apoE plays a direct role in the pathogenesis of the disease. In the present work we have investigated whether apoE can affect the known spontaneous in vitro formation of amyloid-like fibrils by synthetic A beta analogues using a thioflavine-T assay for fibril formation, electron microscopy and Congo Red staining. Our results show that, under the conditions used, apoE directly promotes amyloid fibril formation, increasing both the rate of fibrillogenesis and the total amount of amyloid formed. ApoE accelerated fibril formation of both wild-type A beta-(1-40) and A beta-(1-40A), an analogue created by the replacement of valine with alanine at residue 18, which alone produces few amyloid-like fibrils. However, apoE produced only a minimal effect on A beta-(1-40Q), found in the Dutch variant of Alzheimer's disease. When recombinant apoE isoforms were used, apoE4 was more efficient than apoE3 at enhancing amyloid formation. These in vitro observations support the hypothesis that apoE acts as a pathological chaperone, promoting the beta-pleated-sheet conformation of soluble A beta into amyloid fibres, and provide a possible explanation for the association of the apoE4 genetic isoform with Alzheimer's disease.

The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors.

Abstract: Research Communications Lack of association of G-protein f2and y2-subunit N-terminal fragments provides evidence against the coiled-coil model of subunit-fy assembly Differential calcium dependence in the activity of c-Jun kinase and mitogen-activated protein kinase by muscarinic acetylcholine receptors in rat la cells Evaluation of the use of the luciferase-reporter-gene system for gene-regulation studies involving cyclic AMP-elevating agents

Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3-L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinase.

Abstract: We have investigated the involvement of phosphoinositide (PI) 3-kinase and p70 ribosomal protein-S6 kinase (p70s6k) in mediating insulin stimulation of glycogen synthesis in 3T3-L1 adipocytes using specific inhibitors. Wortmannin inhibited PI 3-kinase activity (IC50 approximately 10 nM), inhibition being complete at 100 nm. Wortmannin (100 nM) completely blocked the ability of insulin to activate glycogen synthase in 3T3-L1 adipocytes and the ability of insulin to stimulate glucose incorporation into glycogen in 3T3-L1 fibroblasts. Rapamycin, which blocks insulin-stimulated activation of p70s6k, decreased insulin activation of glycogen synthase in a dose-dependent manner (IC50 approximately 0.8 ng/ml), with a maximum approx. 75% inhibition of insulin's stimulatory effect. Rapamycin inhibited insulin-stimulated glucose incorporation into glycogen to a similar extent and with similar dose-dependency, while having no effect on insulin-stimulated glucose transport. We conclude that PI 3-kinase and p70s6k are involved in the signalling pathways by which insulin stimulates glycogen synthase in 3T3-L1 adipocytes.

Prediction of O-glycosylation of mammalian proteins: specificity patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

Abstract: The specificity of the enzyme(s) catalysing the covalent link between the hydroxyl side chains of serine or threonine and the sugar moiety N-acetylgalactosamine (GalNAc) is unknown. Pattern recognition by artificial neural networks and weight matrix algorithms was performed to determine the exact position of in vivo O-linked GalNAc-glycosylated serine and threonine residues from the primary sequence exclusively. The acceptor sequence context for O-glycosylation of serine was found to differ from that of threonine and the two types were therefore treated separately. The context of the sites showed a high abundance of proline, serine and threonine extending far beyond the previously reported region covering positions -4 through +4 relative to the glycosylated residue. The O-glycosylation sites were found to cluster and to have a high abundance in the N-terminal part of the protein. The sites were also found to have an increased preference for three different classes of beta-turns. No simple consensus-like rule could be deduced for the complex glycosylation sequence acceptor patterns. The neural networks were trained on the hitherto largest data material consisting of 48 carefully examined mammalian glycoproteins comprising 264 O-glycosylation sites. For detection neural network algorithms were much more reliable than weight matrices. The networks correctly found 60-95% of the O-glycosylated serine/threonine residues and 88-97% of the non-glycosylated residues in two independent test sets of known glycoproteins. A computer server using E-mail for prediction of O-glycosylation sites has been implemented and made publicly available. The Internet address is NetOglyc@cbs.dtu.dk.

Isothiocyanates as substrates for human glutathione transferases : structure-activity studies

Abstract: The catalytic properties of four human glutathione transferases (GSTs), A1-1, M1-1, M4-4 and P1-1, were examined with 14 isothiocyanate (R-NCS) substrates. The compounds include aliphatic and aromatic homologues, some of which are natural constituents of human food, namely sulphoraphane [1-isothiocyanato-4-(methylsulphinyl)butane], erucin [1-isothiocyanato-4-(methylthio)butane], erysolin [1-isothiocyanato-4-(methylsulphonyl)butane], benzyl-NCS, phenethyl-NCS and allyl-NCS. All isothiocyanates investigated were substrates for the four GSTs. The enzymes promote addition of the thiol group of GSH to the electrophilic central carbon of the isothiocyanate group to form dithiocarbamates [R-NH-C(=S)-SG] which have high UV absorption at 274 nm. Molar absorption coefficients and non-enzymic rate constants as well as standardized enzyme assay conditions for all compounds were established. Of the four isoenzymes investigated, GSTs M1-1 and P1-1 were generally the most efficient catalysts, whereas GST M4-4 was the least efficient. Isothiocyanates are among the GST substrates that are most rapidly conjugated. On the basis of rate-enhancement data and binding energies, the isothiocyanates were compared with 4-hydroxyalkenals, another class of natural GST substrates previously subjected to systematic kinetic analysis. The incremental transition-state stabilization attributable to an increased number of methylene groups in homologous alkyl isothiocyanates is similar to that previously noted for homologous 4-hydroxyalkenals.

Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide

Abstract: The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro.

Abstract: Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.

Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes

Abstract: 1. The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides. The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3' terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic. Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides. Oligonucleotides comprised of 2'-methoxy-, 2'-fluoro- or 2'-propoxy-nucleosides did not support RNase H1 activity. 2. The Km and Vmax. of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy 'gapmers', i.e.; oligonucleotides with 2'-methoxy wings surrounding a deoxynucleotide centre. Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency. The initial rates of cleavage increased as the deoxynucleotide gap size was increased. Multiple deoxynucleotide gaps increased the Vmax. but had little effect on Km. 3. The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied.

Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation.

Abstract: The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

Protein hydroperoxides can give rise to reactive free radicals.

Abstract: Proteins damaged by free-radical-generating systems in the presence of oxygen yield relatively long-lived protein hydroperoxides These hydroperoxides have been shown by epr spectroscopy to be readily degraded to reactive free radicals on reaction with iron(II) complexes Comparison of the observed spectra with those obtained with free amino acid hydroperoxides had allowed identification of some of the protein-derived radical species (including a number of carbon-centred radicals, alkoxyl radicals and a species believed to be the CO2 radical anion) and the elucidation of novel fragmentation and rearrangement processes involving amino acid side chains In particular, degradation of hydroperoxide functions on the side chain of glutamic acid is shown to result in decarboxylation at the side-chain carboxy group via the formation of the CO2 radical anion; the generation of an identical radical from hydroperoxide groups on proteins suggests that a similar process occurs with these molecules In a number of cases these fragmentation and rearrangement reactions give rise to further reactive free radicals (R, O2-/HO2, CO2-) which may act as chain-carrying species in protein oxidations These studies suggest that protein hydroperoxides are capable of initiating further radical chain reactions both intra- and inter-molecularly, and provide information on some of the fundamental mechanisms of protein alteration and side-chain fragmentation

Autocrine regulation of milk secretion by a protein in milk

Abstract: Frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. Screening of goat's milk proteins in rabbit mammary explant cultures identified a single whey protein of M(r) 7600 able to inhibit synthesis of milk constituents. The active whey protein, which we term FIL (Feedback inhibitor of Lactation), also decreased milk secretion temporarily when introduced into a mammary gland of lactating goats. FIL was synthesized by primary cultures of goat mammary epithelial cells, and was secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by this milk protein.